Hypoxia (oxygen deprivation) causes metabolic disturbances at physiological, biochemical and genetic levels and results in decreased plant growth and development. Phospholipase D (PLD)-mediated signaling was reported for abiotic and biotic stress signaling events in plants. To investigate the participatory role of PLDs also in hypoxia signaling, we used wild type of Arabidopsis thaliana and 10 pld isoform mutants containing C2-domain. Hypoxia-induced changes in three major signaling players, namely, cytosolic free calcium (Ca-cyt(2+)), reactive oxygen species (ROS) and phosphatidic acid (PA), were determined in mesophyll protoplasts. The Ca-cyt(2+) and ROS levels were monitored by fluorescence microscopy and confocal imaging, while PA levels were quantified by an enzymatic method. Our findings reveal that the elevations of cytosolic calcium and PA are reduced in all the 10 mutants dysfunctional in PLD isoforms. The hypoxia-related changes in both calcium and ROS show different kinetic patterns depending on the type of PLD studied. Pharmacological experiments confirm that both external and internal sources contribute to calcium and ROS accumulation under hypoxia. PLD alpha 1-3, PLD beta 1 and PLD gamma 1-3 are likely involved in calcium signaling under hypoxia as well as in PA production, while all investigated PLDs, except for PLD gamma 3, take part in ROS elevation.

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane-cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLD1 and PLD2 uniquely possess Ca2+-independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pld1 and pld2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia-induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca-cyt.(2+)], hydrogen peroxide (H2O2) and PA. The concentrations of cytosolic Ca2+ and H2O2 were determined by fluorescence microscopy and the fluorescent dyes Fura 2-AM and CM-H(2)DCFDA, specific for calcium and H2O2, respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLD1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLD2 is involved in cytosolic Ca2+ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pld1, while the PA level is unchanged in pld2. Thus, it is likely that AtPLD2 is involved in PA production by a calcium signaling pathway, while AtPLD1 is more important in ROS signaling.